Psychopharmacologically active peptides related to acth

ABSTRACT

The invention relates to psychopharmacologically active peptides with the general formula:

ilnite Greven States tet 1 PSYCHOPHARMACOLOGHCALLY ACTIVE PEPTIDESRELATED TO-ACTH [75] Inventor: Hendrik Marie Greven, Heesch,

Netherlands [73] Assignee: Akzona Incorporated, Ashville, NC.

[22] Filed: Feb. 12, 1973 21 Appl. No.: 331,945

Primary ExaminerLewis Gotts Assistant Examiner-Reginald J. SuyatAttorney, Agent, or FirmFrancis W, Young; Philip M. Pippenger; Hugo E.Weisberger [57] ABSTRACT The invention relates topsychopharmacologically ac- Dec it), 1974 tive peptides with the generalformula:

in which A represents: H.DMet, H-L-Met O), HDMet O), H-LMet 02), H-DMet0 desamino-Met, desamino- Met O), desamino-Met 0 or the moiety H NBCO-,in which B stands for a branched or unbranched alkylene group with 1-6carbon atoms, and in which X represents a hydroxy group, a(N-phenylalkyl)amino group or the group LPheY, in which Y represents ahydroxy group, a (N-aminoalkyl)amino group or the group LLysZ or L-ArgZ,in which Z means a Y PZSX .eteua.-. the r u LL. B.., LTrpG1yOH or a(N3-indoly1alkyl) amino group,

as well as to the functional derivatives of these peptides.

These compounds inhibit the extinction of the conditioned avoidanceresponse, that means that they can be used, in general, asantidepressant agents.

More particularly they can be used for the treatment of certain mentaldisorders whereby a stimulation of the mental performance is desired,such as in certain types of neurosis and in old age infirmities(senility).

7 Claims, No Drawings PSYCHOPHARMACOLOGICALLY ACTIVE PEPTIDES RELATED TOACTH The present invention relates to psychopharmacologically activepeptides and peptide derivatives.

From European Journal of Pharmacology 2, 14 (1967) certain peptidefragments of the natural adrenocorticotrophic hormones (ACTH) are knownto inhibit the extinction of the conditioned avoidance response.Especially the peptide having the amino acid,

sequence l-lO of ACTH proved to be active in this respect. Moreover itwas found that the first three amino acids (SerTyrSer) could evenentirely be omitted without much loss of activity. The article ends withthe conclusion that a peptide with the amino acid sequence 4-10 of ACTHis the shortest peptide, and perhaps the key sequence for the saidactivity.

However, the peptide with the amino acid sequence 4-10 of ACTH does notonly exert the psychopharmacological property, mentioned above, but alsoa slight MSH activity, as usual in this type of fragments of ACTH.Although the effect of a low dosage of an MSH active peptide in men isstill unknown, a search was made for peptideshaving the samepsychopharmacological activity, but no or at least reduced MSl-lactivity.

in first instance it was found in our laboratories, that 4-10 ACTH isnot the key sequence for the said psychopharmacological activity, but amuch smaller peptide having the amino acid sequence 4-6 ACTH.

The peptide HLMet-LGlu-LHis-OH proved to be the shortest peptideinhibiting the extinction of the conditioned avoidance response atpractically the same level as the known 4-10 ACTH, while the tri-peptidedoes not possess a demonstrable MSH activity.

Extension of the chain length at the C-terminal side of the tri-peptidewith Phe-OH (4-7 ACTH), -Phe- Lys(or Arg)OH (4-8-ACT H), -PheLys(orArg)Trp-OH (4-9 ACTH), or -Phe-Lys(or Arg)Trp--Gly-OH (4-10 ACTH) turnedout to cause no marked increase of activity.

It was further observed that the C-terminal aminoacid residues of the4-7, 4-8 and 4-9 ACTH peptides could be replaced by groups which arechiefly distinguished from the said C-terminal amino acid radicals bythe absence of the carboxyl group, without a marked decrease ofactivity.

Thus, for example, the C-terminal amino acid residue Phe in thetetrapeptide 4-7 ACTH (Met- GluHisPhe) could be replaced by a (N-phenylalkyl) amino group of the general formula:

in which Alk represents a branched or unbranched alkylene group with 1-6carbon atoms, and R hydrogen, halogen or hydroxy, or an alkyl or alkoxygroup with 1-4 carbon atoms.

The C-terminal amino acid residue Lys or Arg in the pentapeptide 4-8ACTH (MetGluHis-Phe-Lys or -Arg) could be replaced by a(N-aminoalkyl)amino group of the general formula:

in which Alk represents a branched or unbranched alkylene group with 2-6carbon atoms, R hydrogen or a lower alkyl group, and R hydrogen, a loweralkyl group or an amidine group.

The C-terminal amino acid residue Trp in the hexapeptide 4-9 ACTH(Met-Glu-His-Phe-Lys (or Arg)Trp) could be replaced byv a (N-3-indolylalkyl)amino group of the general formula:

in which Alk" represents a branched or unbranched alkylene group withl-6 carbon atoms. The latter modification appeared to be particularlyvaluable.

Surprisingly it has now been found that the N- terminal amino acid(L-Met) in the above-mentioned peptides and peptide derivatives, thatwas thought to be essential for psychopharmacological activity, can bereplaced by various other groups, which groups do not affect thebiological activity of the peptides in question, but even cause incertain cases a considerable increase of activity.

It is the subject of this invention to modify the N- terminal amino acidresidue L-Met of the key-peptide 4-6 ACTH and of the (at the C-terminalside) extended peptides and peptide derivatives mentioned above into thefollowing groups:

D-methionyl, L- or D-methionylsulfoxide(Met O), L- orD-methionylsulfone(Met 0 desaminomethionyl or the correspondingsulfoxide or sulfone thereof or the moiety encompasses amino acidresidues as well as aminosubstituted carboxylic acid residues, such asGly, Val,

Ala, ,B-Ala and (a-Me)Ala.

From merely synthetic-chemical considerations, the peptides in whichL-Met has been replaced by one of the above-mentioned groupings, thathave no asymetric .centre, such as desamino-methionyl,desamino-methionyl-sulfone, Gly or B-Ala, are preferred because thesepeptides can be prepared more conveniently.

Apart from these synthetic-chemical considerations the modifications ofL-Met into methionyl-sulfoxide (L- or D-Met O), methionyl-sulfone (L- orD- Met 0 desaminomethionyl, desaminomethionylsulfoxide,desaminomethionylsulfone and B-Ala give a considerable increase ofactivity in comparison with the corresponding L-Met peptide, so that, inparticular, these modifications are preferred.

Summarizing, it is the subject of the present invention to provide newand valuable psychopharmacological peptides or peptide derivatives withthe gen-.

eral formula:

A-LGlu-L-His-X in which A represents H-D-Met,. HL-

ll C

(in which R stands for hydrogen, halogen, hydroxy, alkyl (1-4 C) oralkoxy (1-4 C) and Alk" means a branched or unbranched alkylene groupwith 1-6 carbon atoms) or the group LPheY, in which Y represents ahydroxyl group, a (N-aminoalkyl) amino group with the general formula:NHAlkNR R (in which Alk stands for a branched or unbranched alkylenegroup with 2-6 carbon atoms, R for hydrogen or a lower alkyl group (1-6C) and R for hydrogen, a lower alkyl 1-6 C) or an amidine group) or thegroup L-Lys-Z or LArgZ, in which 2 represents a hydroxyl group, thegroup L-TrpOH, L-Trp-- GlyOl-l or a (N-3-indolylalkyl)amino group, thealkyl group of which contains 1-6 carbon atoms,

as well as functional derivatives of these peptides and peptidederivatives thereof.

The peptides and peptide derivatives according to the invention areprepared by a process commonly used in peptid-chemistry. The processesthat are employed usually for the manufacture of the present compoundscan be summarized as follows:

a. condensation of a compound (amino acid, peptide) having a freecarboxyl group and protected other reactive groups, with a compound(amino acid, peptide or amine) having a free amino group and protectedother reactive groups, in the presence of a condensation agent;

b. condensation of a compound (amino acid, peptide) having an activatedcarboxyl group and optionally protected other reactive groups, with acompound (amino acid, peptide, amine) having a free NH group andprotected other reactive groups:

c, condensation of a compound (amino acid, peptide) having a freecarboxyl group and protected other reactive groups, with a compound(amino acid, peptide, amine) having an activated amino group andoptionally protected other reactive groups,

after which the protecting groups are removed, if necessary.

Activation of the carboxyl group can be effected, for example, byconverting the carboxyl group into an acid halide, an azide, anhydride,imidazolide, or an activated ester such as the N-hydroxy-succinimideester, or the p-nitro-phenyl ester.

The amino group can be activated by converting it into a phosphite amideor by the phosphor-azo" method.

Methods usually employed for the above condensation reactions are: thecarbodiimide method, the azide method, the mixed anhydride method andthe method of the activated esters as described in The Peptides," vol.I, 1965 (Acad. Press), by E. Schr'oder and K. L'ubke. MoreoverMerrifields so-called Solid Phase method, described in J. Am. Chem. Soc.85, 2149 1963), can be applied for the manufacture of the presentpeptides and peptide derivatives.

The reactive groups that are not allowed to participate in thecondensation reaction are protected effectiveiy by the socalledprotecting groups, which can be easily removed again, for example, byhydrolysis or reduction. Thus, for example, a carboxyl group can beprotected effectively by esterification with methanol, ethanol, tertiarybutanol, benzylalcohol or pnitrobenzylalcohol, or by conversion into anamide. This latter protecting group is very hard to remove, however, sothat it is recommendable to use this group only to protect the carboxylgroup of the C-terminal amino acid in the ultimate peptide or theY-carboxyl group of glutamic acid. In this case the peptide synthesisleads direct to the amide of a peptide according to formula 1.

Groups that are capable of protecting an amino group effectively areusually acid groups. for example an acid group derived from analiphatic, aromatic, oraliphatic or heterocyclic carhoxylic acid, suchas acetic acid, benzoic acid, or pyridine-carboxylic acid, or an acidgroup derived from carbonic acid such as the group ethoxy-carbonyl,benzyloxy-carbonyl, butyloxy-carbonyl or p-methyloxy-benzyloxy-carbonyl,or an acid group derived from a sulfonic acid. such as the groupbenzene-sulfonyl or p-toluene-sulfonyl, but also other groups can beemployed, such as substituted or unsubstituted aryl or aralkyl groups,for example benzyl and triphenylmethyl, or groups such asortho-nitro-phenyl-sulfenyl and Z-benzoyll methylvinyl.

It is mostly recommendable also to protect the guani dine group ofarginine, the e-amino group of lysine, and the imidazole group ofhistidine, but this protection is not absolutely necessary. Conventionalprotecting groups in this connection are a tertiary butyloxycarbonyl, ora tosyl group for the e-amino group of lysine, a nitro group for theguanidine group of arginine, and a benzyl, dinitro-phenyl or a tritylgroup for the imidazole group of histidine.

The protecting groups can be split off by various conventional methods,depending upon the nature of the group in question, for example withtrifluoro acetic acid, or by mild reduction, for example with hydrogenand a catalyst, such as palladium, or with HBr in glacial acetic acid.

Peptides according to the present invention having as the N-terminalmoiety a methionylsulfoxide or desaminomethionylsulfoxide group, may beprepared from the corresponding Metor Desamino-Met peptide by means of amild oxidation known per se, for example with dilute hydrogenperoxide ora peracid. Such an oxidation yields a mixture of the S- and R-sulfoxide1- or d-sulfoxide), which mixture may be split off into the separatediastereomeric compounds in a conventional manner.

By coupling the S- or R-sulfoxide 1- or dsulfoxide) of methionine ordesaminomethionine with the peptide Glu-His-X, in which X has themeanings indicated above, the separate enantiomers can also be obtainedin a direct way.

The peptides according to the invention having as the N-terminal residuea methionylsulfone (Met 0 or desaminomethionylsulfone (desamino-Met 0group may be prepared most conveniently by an oxida tion known per se ofthe corresponding Metor Desamino-Met peptide, for example with H 0 or aperacid.

By functional derivatives of the peptides and peptidederivativesaccording to the invention are meant:

I. the pharmaceutically acceptable acid addition salts of the peptidesand peptide derivatives,

2. peptides or peptide derivatives in which one or more free aminogroups have been substituted by an acyl group derived from an aliphaticcarboxylic acid with 1-6 carbon atoms, such as an acetyl group,

3. unsubstituted amides or lower alkyl (l-6 C) substituted amides ofthose peptides and peptide derivatives according to the invention havinga free carboxyl group, p

4. esters of the present peptides derived from aliphatic or araliphaticalcohols with 1-18 carbon atoms, such as methanol, ethanol, pentanol,hexanol, cyclohexanol, octylalcohol, undecylalcohol, hexadecylalcohol,oleylalcohol, octadecylalcohol, benzylalcohol, phenylethylalcohol,phenyl propylalcohol, or cinnamylalcohol,

5. metal complexes formed by contacting the peptides or peptidederivatives with a sparingly soluble salt, hydroxide or oxide of ametal, preferably zinc, or preparations obtained by associating thepresent peptides with organic, mostly polymeric, compounds, such asgelatine, polyphloretinphosphate or polyglutamic acid, to obtain aprolonged mode of action.

The acid addition salts are obtained by reacting the present compoundswith a pharmaceutically acceptable organic or inorganic acid, such asl-lCl, phosphoric acid, acetic acid, maleic acid, tartaric acid orcitric acid.

As already briefly said the present peptides and peptide derivatives aswell as their functional derivatives defined above have valuablepsychopharmacological activities. The present compounds inhibit theextinction of conditioned avoidance response, that means that they canbe used, in general, as antidepressant agents. More particularly theycan be used for the treatment of certain mental disorders whereby astimulation of the mental performance is desired, such as in certaintypes of neurosis and in old-age infirmities (senilit Tli e peptidesaccording to the invention and the functional derivatives defined abovecan be administered orally, parenterally or intranasally. Preferably thepeptides are employed as an injection preparation, for

which purpose they are dissolved, suspended or emulsified in a suitableliquid, but mixed with suitable auxiliaries and fillers they can also beplaced in a form suitable for oral administration, such as pills,tablets or coated tablets. The present peptides can also be administeredin the form of suppositories or sprays.

The peptides or peptide derivatives according to the invention arepreferably administered in daily dosages of from 0.001 to 1 mg per kgbody weight, dependent upon the peptids activity level and theform inwhich they are administered. 5 Exceedingly valuable preparations 'areobtained if the present peptides are placed in a form in which they havea prolonged activity, for example, incorporated into gelatin,polyphloretinphosphate or polyglutamic acid, or preferably as metalcomplexes. These metal complexes can be obtained by contacting thepeptides with sparingly soluble metal salts, metal hydroxides or metaloxides. As sparingly soluble metal salts the metal phosphates, metalpyrophosphates and metal polyphosphates are commonly used.

Metals than can be used in this process are the metals belonging to theb-groups of the periodic system, for example cobalt, nickel, copper,iron, and preferably zinc, as well as the metals belonging to the maingroups of the periodic system and capable of fonning complexes, such asmagnesium and'aluminium. The preparation of the said metal complexestakes place in the conventional manner.

Thus, for example, a metal complex can be obtained by adding the peptideand a poorly soluble metal salt, metal hydroxide or metal oxide to anaqueous medium. The metal complex can also be obtained by adding analkaline medium to an aqueous solution of the peptide and an insolublemetal salt to form the insoluble peptide/metal hydroxide complex.

Moreover, the metal complex can be obtained by adding the peptide, asoluble metal salt and a soluble salt to an aqueous, preferably alkalinemedium to form an insoluble peptide/metal salt complex in situ.

The metal complexes can be employed at once as suspensions, or forexample be lyophilized and afterwards suspended again. Biologicalactivity: Extinction of the conditioned avoidance response.

Male white rats weighing approximately 150 grams were conditioned bymeans of the so-called polejumping test. The conditioned stimulus was alight presented over the cage for 5 seconds, whereupon the unconditionedstimulus of shock was delivered through the grid floor of the cage.

For 3 consecutive days 10 tests were run every day with an averageinterval of 60 seconds. The day after this acquisition period theextinction was studied in sessions of 10 trials. All animals that made 8or more positive responses in the first extinction session were treatedwith the substance to be tested or with a placebo. After that,extinction sessions of l0trials each were carried out 2 and 4 hoursafter the treatment of the animals with the substance to be tested.

In the following table the results of the known peptide 4-1O ACTH arecompared with some peptides according to this invention.

Peptide or peptide Dosage Estimated derivative in 1.1.gm First SecondThird potency per session session session ratio animal after after (4-10ACTH sc. 0 hour 2 hrs. 4 hrs. =1)

i cii gi-i 9 s 7 g rp y 30 9 7 5 l0 9 6 3 l H D Met-Glu-His OH 30 8 6 5l H Val Glu-His-OH 30 8 6 4 l IV. For the amino acid residues thefollowing abbreviations have been used:

Met methionyl Met( O) methionylsulfoxide (racemic) Peptide or peptideDosage Estimated derivative in fLgTTl First Second Third potency persession session session ratio animal after after (4-10 ACTH s.c. hour 2hrs, 4 hrs. ==1

H-fi-AIa-GIu-His'OH 3O 9 8 7 1O 9 7 6 3 H-Met( O)-Giu-His- 3O 8 8 8 OH10 8 8 7 3 8 4 2 3 H-D-Met-G1u-His- Phe-OH 3O 8 7 7 1O 8 6 2 1H-D-Met-Glu His Phe-Lys-OH 3O 8 7 5 1 H-D-Met-Glu-His- PheLys-Tra 3O 1O1 O 8 1O 8 8 7 3 9 8 7 H-Met( O)-Glu-His- Phe-Arg-Trp-Gly-OH 10 8 8 8 38 4 2 3 H-Met(d, O)-Glu-His- Phe-Arg-Trp-Gly-OH 5 9 7 6 1.5 9 7 3 6H-B-Ala-Glu-His-Phc- ArgTrp-Gly-OH 9 8 8 1O 9 8 6 3 H-D-Met-Glu-His-Amf3O 8 8 (i 10 9' 7 3 1 Desamino-Met-Glu-His- Phe-Lys-Trp-OH 30 9 s s 10 97 6 3 9 5 3 3 Desamino-Met( O Glu-His-Phe-Lys-Trp- OH 10 8 3 8 3 9 6 5 18 5 2 10 6th regard to the various abbreviations used Met(c1, 0)methionyitdkulfoxide throughout the specification, examples and claimsthe Mam-"0) l 'l Met(- O methionylsulfone following remarks are made: 61glut-am 1. If no optical configuration has been stated the L- 0TGluiNH-il jfi g g y form is meant. p phenyialanyi II. The followingabbreviations have been used for if"; j s y the protecting or activatinggroups: fi gg Gly glycvl 31 =glalyl z =benzyloxycarboiiyl Boc =tert arybutyloxycarbonyl E3 2 Am & 2 23 alan I tBu tertiary butyl y 3413? hp-nitro enyloxy 4 ONE p-nitro nzyloxy 5 Su =succinimido V. Abbreviationsfor other residues:

Ill. For the solvents or reagents the following abbrev viations havebeen used: Tm gggzzggg h group (derived from Desamiiiodesamino-methionyl(or 'y-methylthiobutyryl) Met group B1 benzene Amf=(N-1-pheny11sopropyharruno group (derived 0 ethanol from amfetarnine).Bu butariol Py pyridine Ac or HAc acetic acid EXAWLE I Wa water $1 f gst Eggs A. HMet( O)(iluHis-Ol-l i di z flg lf id 25 mg of the tripeptidel-l-Met-Glu-l-lm-OH are THF l'qtmhydrofuwn dissolved in 2.5 ml of aceticacid, after which 15 .Ll of gggfifif$f 30 percent hydrogen peroxide areadded. After stirring TAA tri-ethylamine for 1 hour at 20 C, asuspension of 20 mg of platinum TFA trifluoro acetic acid black in 2.5ml of glacial acetic acid is added and the mixture is stirred for 30minutes. Then the mixture is filtered and the solvent distilled off invacuum. The resulting residue is taken up in 10 ml of tertiary butanoU-water (1:1 and lyophilised. Rf of the sulfoxide 0.14 (4:3/4:1f4:1) onSiO in Bu:Py:Ac:Wa

EXAMPLE 11 and the pH is adjusted to 6.9 with triethylamine. Stirring iscontinued for 3 days at after which the mixture is filtered and thefiltrate evaporated to dryness in vacuum. The residue is taken up in 150ml of ethyl acetate/water and washed with water. The organic phase isdried, evaporated to 100 ml and set aside at 0. Rf in BuzAczWa (4:121)0.63 (SiO Melting point: 697 1C. B. DesaminoMet-Glu(OtBu)-His-OMe 1n thesame manner as described in HA, the hydrazide desaminoMetN H isconverted into the azide and then coupled to H-Glu(OtBu)-His-OMe.2 HCl,whereafter the compound obtained is isolated and purified as indicatedin A. Rf in Bu:Ac:Wa (4:1:1) 0.50 on SiO C. BocDMetGlu(OtBu)His-OHTripeptide ester (1.17 g; example ll.A) is dissolved in 50 ml of 50percent dioxane. To this solution 2.5 ml of l N sodium hydroxide areadded, after which the mixture is stirred for 30 minutes at 20 C. Thereaction mixture is neutralised to pH 7, after which it is evaporated todryness in vacuum. The residue is taken up in 40 ml of ethyl acetate,acidified with 1.4 ml of 2 N HCl to pH 4 and washed with a little water,after which the layers are separated.

The ethyl acetate layer is dried and evaporated to dryness. The residueis an oil. Rf in Am:Py:Wa (5:312) =0.l9 (SiO D. BocDMetGlu(NHQ-His-OH 1gram of tripeptide ester is dissolved, while heating, in 20 ml ofdioxane/water (1:1). The mixture is cooled down to 20 C, after which 1.9milli equiv. of barium hydroxide are added and the mixture is stirredfor 30 minutes. After neutralisation to pH 5.4, the mixture is carefullyevaporated. The residue is dissolved in methanol and filtered, and thefiltrate diluted with ethyl acetate.

The precipitate is filtered and recrystallised from methanol/ethylacetate. Rf in Am:iPro:Wa (10:4:5) 0.59 (SiO E.BocD-Met'-Glu(OtBu)HisNl-l 1n a cooled solution of dry ammonia gas in 25ml of methanol 1 g of BocD-Met-Glu(0t- Bu)His-OMe is dissolved (example11A). The mixture is stirred for 25 hours, after which the ammonia isevaporated, the residue taken up in methanol and the solution dilutedwith ether. The precipitate formed is filtered off. Melting point: 72-75C. Rf in BzzEtOH (8:2) 0.43 (SiO F. DesaminoMet-Glu(OtBu)-HisOH Theester obtained in 11B is hydrolysed in the same manner as described inll.C. Rf in AmzPyzWa (513:2) =0.l6 (SiO G. Removal of protectinggroup(s) from the peptides obtained in A-F 100 mg of the peptide (A, B,C, D, E or F) are dissolved in 5 ml of 90 percent TFA. The solution isleft to stand for 1 hour, after which the solvent is distilled off, theresidue stirred twice with ether and dried over solid potassiumhydroxideQThe trifluoro acetate is dissolved in 20 ml of t-butanol/water(1:1), after which it is converted into acetate by stirring the solutionwith so much Dowex X-8 inthe acetate fonn to obtain a final pH of 5.4.After filtration the filtrate is lyophilisedand the .white residuestored over solid phosphor pentoxide.

H. Sulfoxide of HDMet-GluHis-OH 25 mg of the tripeptide HD-Met-Glu--His-OH (G. 2) are dissolved in 2.5 ml of acetic acid, afterwhich 15 pl of 30 hydrogen peroxide are added. The mixture is treated inthe manner described in example 1. The residue is then taken up in 10 mlof t-butanol/water- (1:1), after which this solution is lyophilised. Rfin Bu:Py:Ac:Wa (4:3/4:1/4:1) =0.l2 on SiO K. Sulfoxide ofdesamino-MetGlul-lis-OH 25 mg of the peptide obtained in G. 4 isoxidized in the same manner as described in H. Rf in Bu:Py:Ac:Wa(4:3/4z1/4zl) =0.l8 on SiO EXAMPLE 111 Synthesis of A-Glu-Hisderivatives (A=Val, Gly,

Ala, ,B-Ala-or (a-Me)Ala) A. BocV al-Glu(OtBu)-His OMe Boc-VaI-OH (3.26g; 15 mmol) is dissolved'in 20 ml of methylene chloride, after which1.73 g of N- 7 hydroxy succinimide are added. The solution is cooleddown to 20 C, after which 3.09 g of DCCl, dissolved in 20 ml of cooledmethylene chloride are added and the resulting solution is stirred for 1hour at 20 C and then for 20 hours at +20C.

The resulting DCHU is filtered, after which the filtrate is evaporatedto dryness and the residue dissolved in 30 ml of DMF, whereupon 7.33 gof Z-G1u(Ot- Bu)-His- -OMe (prepared in accordance with Kappler Helv.44, 1991, 1961) and 1.4 g of 10 percent pal ladium/carbon are added.Then hydrogen is bubbled through for 5 hours, after which the mixture isstirred for 1 night and filtered, and the filtrate evaporated todryness. The residue is dissolved in aqueous ethyl ace tate and washedwith citric acid, water, sodium bicarbonate and water. The organic phaseis dried, after which the ethyl acetate is evaporated in vacuum. Theresidue is recrystallised from ethyl acetatepetroleumether. Yield 3.95g; melting point: 1 l7-119 C; [011 =8.4 (0 =1 DMF); Rf in BzzEtOH (8.2)0.55 (SiO BocGly--Glu(OtBu)l-lis-OMe Starting from 2.63 g of Boo-Gly-OHthe tripeptide Boc-Gly-Glu (OtBu)His-OMe is prepared by the processdescribed in example A. Yield: 1.47 g: melting point: l03-108 C; [0415.2 (C 2, DMF). Rf in Bz:EtOl-l (8:2) =0.43 (SiO C.BocAla--Glu(OtBu)l-lisOMe Boc-AlaOl-l (3.78 g) is converted into theactive ester with 2.3 g of N-hydroxy-succinimide and 4.12 g of DCCI. Theactive ester, dissolved in 40 ml of DMF, is condensed withH-Glu(OtBu)--His-Ol\ le, obtained from 9.77 g of ZGlu(OtBu)HisOMe asdescribed in example [H.A. Yield: 5.56 g; melting point: 97.5-101 C. Rfin Bz:EtOl-l (8:2) =0.36 (SiO D. BocBAla-Glu(OtBu )HisOMe Starting from3.78 g of BocBAlaOH (melting point 777 8 C) the peptide ester BocB-AlaGlu(OtBu)His-OMe is prepared in the same manner as described in examplelll.C. Yield: 7.4 g; [621922 8.7 (c= 2, DMF). Rf in BzrEtOH (8:2) 0.25(SiO E. Boc-(a-lvle)AlaGlu(OtBu)His-OMe Boc( l-methyl)alanine (1.83 g;m.p. 1181-l19.5 C) is dissolved in 40 ml of methylene chloride, afterwhich 3.51 g of H-Glu(OtBu)l-lis-OMe.2 HCl, prepared by hydrogenation ofthe Z-peptide in methanol, in the presence of 2 equiv. hydrochloricacid, and 2.29 ml of TAA are added to this solution. The mixture iscooled down to 0, after which 2.07 g of N-hydroxysuccinimidc are addedand, finally, after the mixture has been cooled down to 20, 1.86 g ofDCCI. In the manner described before (example A) the reactionmix ture istreated further after being stirred for 10 min at C, for 2 hours at 0 Cand for 20 hours at 20 C. Yield: 3.02 g of oil. Rf in Bu:Ac:Wa (4:l:1)=0.61 (SiO F. Hydrolysis of the peptide esters A, B, C, D and E 1.15 g ofthe peptide ester A, B, C, D or E is dissolved in 50 ml of 50 percentdioxane. After the addition of 2.5 ml of l N sodium hydroxide, themixture is stirred for minutes at 20 C. The reaction mixture isneutralised to pH 7, after which it is evaporated to dryness, theresidue taken up in 40 ml of ethyl acetate, and the solution washed with0.7 ml of 2 N hydrochloric acid (pH 4). The ethyl acetate is dried andevaporated to dryness.

Rf in AmzPyzWa (5:312) (SiO 50 G. Removal of protecting groups of thepeptides obtained in F.1F.5.

In the manner as described in Example [LG the peptides mentioned inlll.F are deprotected with TFA and 60 converted into the acetate.

1-1. Removal of protecting groups from the peptide esters obtained inA-E.

In the manner as described in example 11.6 the tripeptide estersobtained in A, B, C, D and E are do protected and converted into theacetate.

EXAMPLE IV Synthesis of l-l(a--Me)AlaGlu-HisR (R NHz, Or

A. Boc-(o:Me)AlaGlu( OtBu)HisOl\/le (0.7 g; example 111.13) is dissolvedin a cooled solution of dry ammonia gas in 20 ml of methanol. Themixture is stirred for 20 hours, after which the ammonia is evaporated,the residue taken up again in methanol and the solution diluted withether. The precipitate formed is filtered off (0.41 g); m.p. 93-95 C.The precipitate is then dissolved in 20 ml of percent TFA. The solutionis left to stand for 1 hour. after which the solvent is distilled off invacuum, the residue obtained stirred into ether and dried over KOHtablets. The tritluoro acetate is then exchanged for the acetate byadding enough Dowex X-8 in the acetate form to a solution of the peptidein t-butanol/water (1:1) to reach a pH of about 5.4. Filtration of themixture and lyophilisation of the filtrate yields an almost white power,being the pure peptideamide H-(aMe)Ala--Glul-lis-l\ll-l Rf inBu:Py:Ac:Wa (4:3/4:l/4: 1) =0.20 on SiO B. If instead of dry ammoniagas, dimethylamine or ethylamine is used, the correspondingN,N-dimethylpeptide-amide or N-ethyl-peptide-amide is obtained.

EXAMPLE V Synthesis of DMetGluHis-Phe derivatives A.Z-Glu(OtBu)His-Phe-OMe ZGlu(OtBu)HisN l-l (9.76 g) is dissolved in 30 mlof DMF, after which the solution is cooled to 0 C.

After the addition of l4.4 ml of 4.8 N hydrochloric acid/THF, thesolution is cooled further to 20 C, after which 3.24 ml ofisoamyl-nitrite are added dropwise. The solution is stirred for 7minutes, after which it is added to a solution of 4.32 g ofHPheOMe.l-1Cl in 40 ml of DMF and 12.6 ml ofTAA, precooled to 0 C. Themixture is left to stand for 3 days at 0 C, after which it is filtered.The filtrate is taken up in ethyl acetate/water. The organic phase iswashed successively with water, sodium bicarbonate and water, and afterthat dried, whereupon the solvent is distilled off in vacuum. Theresidue is recrystallised from hot ethyl acetate. Melting point: 183,[(21 9 -2l (c 1 DMF). Rf in Bz:EtOl-l (8:2) (SiO 0.81. B.Z-Glu(OtBu)l-lisPhe-OH To an equal quantity of the azide ZGlu(Ot-Bu)-l-lis-N as prepared in A, are added 3.3 g of phenylalanine and 12.6ml of triethylamine, dissolved in 15 ml of DMF of 0 C. The mixture isleft to stand for 3 days at C, after which it is filtered. The filtrateis evaporated to dryness, taken up in ethyl acetate/water, acidifiedwith percent citric acid to pH 3 and then washed with water. The layersare separated, and the organic layer is dried and evaporated.

The residue is recrystallised from acetonitril/ether. Rf in Am:Py:Wa(5:3:2) 0.73 (SiO C. Z-Glu(OtBu)l-lis-(N--phenylethyl)amide Thephenylalanine used in B is replaced by B-phenylethylamine to obtain thepeptide Z-Glu(OtB- u)-His--(N-phenylethyl)amide.

1 gram of the tripeptide ester as described in A is dissolved in 20 mlof methanol, after which ammonia gas is bubbled through the mixture.Then the mixture is stirred for 1 night, after which the resultingprecipitate is filtered. After crystallisation. from methanol/ether theamide is obtained. Melting point: 1l9-l20 C.

Rf in Bu:Py:Ac:Wa (4:3/4z1/411) =0.70 (SiO E. Deprotection of thepeptides or peptide derivatives A-D (incl.)

0.7 g of the peptide obtained in A, B, C or D is dissolved in 18 ml ofpure methanol, after which percent palladium on charcoal is added.Hydrogen is bubbled through the mixture till no CO escapes anymore,after which the mixture is filtered and the filtrate evaporated invacuum at a bath temperature of 30 C. The partially deprotected peptideis immediately processed further. I

F. Condensation of D-Met with peptides prepared in E BocD-MetN H (3.92g) is dissolved in 75 ml of DMF. The solution is cooled down to 0 C,after which 7.5 ml of 4N HCl/T HF are added and the solution is cooleddown further to -20 C. lsoamyl-nitrite (2.1 ml) is added, after whichthe mixture is stirred for 5 minutes and the volume made up to 90 ml(solution Meanwhile 5 mmol of one of the tripeptides prepared in E hasbeen dissolved in 20 ml of DMF and 2 ml of water. This solution iscooled down to 0 C.

Of solution A" 30 ml are added to the tripeptide solution. The mixtureis left to stand for 72 hours at 0 C, after which the DMF is distilledoff and the residue taken up in ethyl acetate/water.

The organic phase is washed with bicarbonate and water, after which theorganic phase is dried and the solvent distilled off in vacuum.

The residue is crystallised from methanol-ether.

Peptide: Rf in BzzEtOH (8:2) on SiO,

1. Boc-D-Met-Glu(OtBu)-His-Phe-OMe 0.42 2.Boc-D-Met-Glu(OtBu)-His-Phe-OH 0.14 2. Boc-D-Met-G1u(OtBu)-l-lis-Phe-NH0.20 0.40

Boc-D-Met-Glu(OtBu)-His-( N phenylethyl amide G. Condensation ofdesamino-Met with the peptides prepared in E In the same manner asdescribed in F, desaminomethionylhydrazide (5 'mmol) is converted 'intothe corresponding azide by means of isoamylnitrite, after which theazide formed is coupled to one of H. Of the peptideBocMet-Glu(OtBu)-His- Phe-OH prepared in E2, 0.25 g is dissolved in 1.4ml

of undecanol and 0.3 g of Dowex X-8.1-1Cl (basic resin). The mixtureisstirred for 7 days at room temperature and filtered, and the filtrateevaporated in vacuum. The undecyl ester is isolated by chromatography ofthe residue obtained over SiO by means of the eluent benzene/ethanol(9:1). Rf in BzzEtOH (8:2)

0.68 (SiO K. Deprotection of the peptides obtained in F, G and H In asimilar manner as described in example 11.6 the peptides obtained in F,G and H are deprotected.

L. Sulfoxide Of the tetrapeptide HMet-Glul-1is.Phe-OH 30 mg aredissolved in 2.5 m1 of acetic acid after which 15 p1 of 30 percent H areadded. The mixture is processed further in the manner described inExample I. The residue is then taken up in ml of t-butanol/water (1:1),after which the mixture is lyophilised.

1n the same manner the peptides obtained in 14.1, K2 and K.7 are alsoconverted into the corresponding sulfoxide.

Example Vl Synthesis of ValGlu-I-lis-Phe derivatives A.BocVal-Glu(OtBu)-HisPhe-NH Boc-Val-Ol-l (0.33 g) is dissolved in ml ofDMF, after which 0.81 g of HGlu(OtBu)I-iis PheNl-l (example V.E.3) isadded and the pH adjusted to 7.2. After the addition of 0.31 g of DCCI,the mixture is stirred for 5 hours at 0 C and for 20 hours at 20 C. Theresulting DCHU is filtered off, after which the filtrate is poured into100 ml of 0.1 N sodium bicarbonate and the precipitate stirred for 1hour at 0 C. After filtration the residue is dried and recrystallisedtwice from ethyl acetate/ether. Yield: 0.51 g; m.p. 137 (dec.). Rf inBz:EtOH (8:2) =0,27 on SiO B. l-l-Val-Glu-His-Phe-Nl-l Deprotection ofthe protected tetrapeptide-amide of A by the process described inexample ll.G yielded the acetate of the above peptide.

EXAMPLE VII Synthesis of D-Met-Glu-His-(N-phenyialkyl)amidesBoc-D-Met-Glu(OtBu)-His-N H (1.17 g; example IX.A) is dissolved in 20 mlof DMF. This solution is cooled down, after which 3 ml of 2 N HClfTHFare added at 0 C and 0.27 ml of isoamyl-nitrite at 20 C, and the mixtureis stirred for 7 minutes at 20 C.

To the azide solution 3 mmol of the required amine are added andadjusted with TAA to pH 7.

The reaction mixture is stirred for 70 hours at 0 C, after which it isevaporated to dryness in vacuum and the residue dissolved in aqueousethyl acetate.

The organic phase is washed with water, bicarbonate and water, anddried.

The residue is evaporated to dryness, then stirred withether/petroleumether, and dried again.

By the process described in example [1.0 the protecting groups areremoved.

"cf. Example V.K.3

EXAMPLE VIII Synthesis of AGluHis--(Nphenylalkyl)amides (A Val 0r B-Ala)A. BocVal-Glu(OtBu)-HisOMe (1.11 g; example llLA) is dissolved in 20 mlof methanol. after which 1 ml of hydrazine hydrate is added. The mixtureis stirred for 6 hours at room temperature, after which the methanol isdistilled off in vacuum, the remaining residue stirred with water andthen dried.

The dry residue is then dissolved in DMF and coupled to i-amphetamine(l-phenylisopropylamine) by the azide method as described in example V11.

In the manner described in example ll.G, the resulting compound isdeprotected to obtain the peptide H- ValGlu-His( N- l -phenylisopropyl)amide .acetate, which peptide is lyophilised.

Rf in Bu:Ac:Wa (411:1) 0.18 on SiO Starting substance 0.53.

B. Boc-B-Ala-Glu(OtBu)His-OMe is converted into the correspondingH-)3AlaGlu-- His-(N-benzyl)amide in the same manner as described in A onthe understanding that instead of l-amphetamine now benzylarnine isused.

Rf in Bu:Ac:Wa (4:1:1) 0.16 on SiO EXAMPLE IX Synthesis ofDMet--GluHis-Phe-Arg A. BocDMet-Glu(OtBu)-HisN l-lBocD--MetGlu(OtBu)l-lisOMe (3.2 g; example ll.A) is dissolved in ml ofmethanol, after which 3.7 m1 of hydrazine hydrate are added. The mixtureis stirred for 5 hours at room temperature, after which the methanol isdistilled off in vacuum and the residue stirred with water.

After being dried, the hydrazide is at once processed further. Rf inAm:iPro:Wa (10:4:5) 0.37 on SiO B. BocDMet--Glu( OtBu )HisPhe-ArgOH Ofthe tripeptide-hydrazide (A; 1.17 g) is dissolved in 20 ml of DMF. Afterthe addition of 3 ml of 2N HCl in THF, at 0 C, the mixture is cooleddown further to -20 C, after which 0.27 ml of isoamyl-nitrite is added.The mixture is stirred for 6 minutes after which the azide solution isadded to 2 mmol of the peptide l-i-Phe Arg-OH in 5 ml of DMF, afterwhich the pH is adjusted to 7.3 with TAA.

The reaction mixture is stirred for 70 hours at 0 C and then evaporated.The residue is stirred into ethyl acetate. Evaporation of this ethylacetate gives an amorphous pentapeptide.

Deprotection of the pentapeptide obtained in B with trifluoro aceticacid yielded the pentapeptidediacetate after the exchange with Dowex X-8in the acetate form.

Rf in Bu:Py:Ac:Wa (423/421/4c1) =0.18 on SiO EXAMPLE X In the mannerdescribed in example 1 the pentapeptides l-l-MetGluHis-PheArgOl-l,H-Met-Glu-HIs-PheLys-OH and 17 l-IDMetGluI-IisPheArgOl-I (example IX.C)are oxidised by means of 30% H in acetic acid.

EXAMPLE XI on SiO yield 79 percent. The dipeptide I-I--,

PheLys(Boc)-OtBu is obtained in 95 percent yield by hydrogenation of thepeptide ZPhe-Lys(Boc- )OtBu in methanol with percent palladium oncharcoal.

Rf in BuzAczWa (411:1) 0.41 on SiO Of the protected peptideBoc-(l--Me)Ala- Glu(OtBu)I-IisOMe (example III.E; 2.9 g) is convertedinto the hydrazide with hydrazine hydrate in the manner described inexample IX.A. melting point: 21 8225 C dec.

C. In the same manner as described in B the methyl esters:BocAla-Glu(OtBu)I-IisOMe, (Example IILC), BocB-Ala-G1u(OtBu)I-IisOMe(Example III.D) and DesaminoMet-Glu(0t- Bu)-HisOMe (Example II.B) areconverted into the corresponding hydrazides. D.l-I-(a--Me)AlaGluHis--PheLys--OH The peptide-hydrazide of B (1 mmol) isdissolved in 10 ml of DMF. The mixture is cooled, after which 3 ml of 1NI-ICl in THE and 0.13 ml of isoamyl-nitrite are added. The mixture isthen stirred for 6 minutes at C. The thus obtained azide is added to asolution of 1 mmol of I-IPheLys(Boc)OtBu (from A) in 5 ml of DMF. Afterthe pH has been adjusted to 7.4 with TAA, the mixture is stirred for 70hours at 0 C. The DMF is distilled off in vacuum, the residue dissolvedin aqueous ethyl acetate and the organic phase washed with water,bicarbonate and water. The ethyl acetate is dried and then distilled offin vacuum. The residue is chromatographed over SiO and the peptide B0c(-aMe)AlaGlu(OtBu)--His--Lys(Boc)--OtBu isolated.

Rf in BzzEtOH (8:2) 0.74 on SiO Deprotection of this protected peptidein the manner described in example ".6, gives the abovepentapeptide.aceta te in 63 percent yield.

Rf in Bu:Py:Ac:Wa (4:3/4:l/4:1) 0.18 on SiO In the same manner asdescribed in Dthe peptide methyl esters obtained in C are converted intothe corresponding azide and coupled with H--Phe-Lys( Boc- )--OtBu (A).

Removal of the protecting groups, according to the manner described inExample II.G, gives the following unprotected peptides as the acetatesin 60-70 percent yield:

Rf in Bu:Py:Ac:V Va (423/4: 1/4: I) on SiO Peptide:

H-B-Ala-Glu-His-Phe-Lys-OH 0. l 8 H-Ala-Glu-His-Phe-Lys-OH 0.20Desamino-MetGlu-His-Phe-L s-OH 0.24 H-D-Met-Glu-His-Phe-LysO 0. l8

EXAMPLE XII Of the tripeptide-hydrazide Boc-DMet-Glu(0t- Bu)I-IisN I-I(example lX.A) 1.17 g are dissolved in 20 ml of DMF. After the additionof 3 ml of 2 N l-ICl in THF, at 0 C, the mixture is cooled further to 20C. Then 0.27 ml of isoarnyl-nitrite is added to this mixture, whereuponthe mixture is stirred for some time. 7

The azide solution obtained is then added to 2 mmol of the compoundI-IPheNH(CI-I N Boc (obtained by hydrogenation of the correspondingcompound Z-Phe-NI-L-(CI-bk-NH-Boc, l3 l-l33 C) in 5 ml of DMF,-afterwhich the pH is adjusted to 7.5 with TAA.

The reaction mixture is stirred for hours at 0 C, after which it isevaporated and the residue stirred into ethyl acetate. After evaporationof the ethyl acetate a yellowish white amorphous powder is obtained,which is treated further in the manner described in example II.G. Aftertreatment with Dowex X-8 in the acetate fomi, theHD-MetGluI-lis--Phe--NI-I-(CH NH .diacetate is obtained,

In the same manner as described (in A) BocGly-- Glu(OtBu)l-IisN H(obtained from the corresponding methylester) is converted into'thediacetate of l-IGlyGlu-I-Iis--PheNH-(CH -NH Rf in BuzPyzAczWa(2:3/4:l/4:l) =0.32 on SiO C. Desamino-Met-GluI-IisPheNH-(CH )s' 2 Inthe same manner as described (in A) Desamino- MetGlu(OtBu)--I-IisN I-I(see Example XLc) is converted into the acetate of Desamino-Met--GluI-IisPheNl-I(CI-I NI-I Rf in Bu:Py:Ac:Wa (2:3/4:l/4:l) =0.38.

. EXAMPLE XIII Synthesis of- ValGlu--HisPheArg-Tra A.Boc-Val--Glu(OtBu)I-Iis-N I-I Starting from the tripeptide ester(example III.A) the hydrazide Boc-Val--Glu(OtBu)-His-N H is obtained inthe manner described in example VIII.A. Rf in AmziProzWa (10:4:5) =0.33on SiO- B. BocArg(NO )Tra Boc--Arg(NO )OH (5.76 g) is dissolved in 70 mlof DMF-and 2.52 ml. of TAA. This solution is cooled to 10 C, after which2,38 ml of isobutyl chloroformate are added and the mixture is stirredfor 10 minutes at l0 C. To this solution is added a solution of 2.9 g oftryptamine in 10 ml of DMF and 2.32 ml of TAA, the temperature beingmaintained at about -10 C The mixture is stirred for 30 minutes at l0 C,for 2 hours at 0 C and for 18 hours at 20 C, after which the solvent isevaporated in vacuum and the residue taken up in ethyl acetate/water.The organic phase is washed with water, bicarbonate (5 percent) andwater, and then dried and evaporated.

Yield 7.4 g of oil; Rf in BzzEtOH (8:2) =O.54 on $10 C. ZPhe-Arg(NO)--Tra and HPhe--AIg-Tra Z PheONP (2.1 g) is dissolved in 10 ml of DMFand 10 ml of ethyl acetate. Then 1.81 g of H-Arg- (NO )Tra (obtained bycleavage of the amide of B in the manner described in example [1.6) areadded, after which the mixture is stirred for 30 minutes at 10 C and for20 hours at 20 C. The reaction mixture is processes as described in B.

Yield: 1.3 g; melting point: 131-l 33 C.

Rf in BzzEtOH (8:2) 0.47 on SiO Of the compound obtained 1.3 g areconverted into l-l-Phe-ArgTra by hydrogenation over palladium 10 percenton charcoal. Yield 72 percent.

The hydrazide prepared in A is converted into the azide by means ofisoamyl-nitrite, in a conventional manner, and coupled to the peptidel-lPheArgtryptamide of C. The reaction mixture is stirred for 70 hoursat C and poured out into 0.1 N sodium bicarbonate to precipitate thepeptide.

After filtration the precipitate is dissolved in DMF and solution pouredout into a tenfold quantity of ethyl acetate. Filtration gives a residuewith Rf in Bu:- PyzAczWa (4:3/421/421) 0.54 on SiO Cleavage in themanner described before (example [1.6) gives the acetate ofH-Val-Glu-His-Phe-Tra in 33 percent yield.

Rf in the same solvent mixture 0.18.

deriva- Rf in BzzEtOl-I (8:2) 0.70 on SiO B. l-lPhe-Lys(Boc)-Tra 2 gramsof the peptide described above (of A) are dissolved in 25 ml of methanoland hydrogenated in a conventional manner, in the presence of 10 percentpalladium/charcoal. After evaporation to dryness a foam is obtained.Yield: 95 percent.

Rf in BuzActWa (411:1) 0.80 on SiO C.BocAGlu(OtBu)-l-1is--Phe-Lys(Boc)-Tra (A Met, D-Met or l3-Ala) Thetryptamide derivative of B in DMF (1.5 g) and the hydrazideBocAGlu(OtBu)-l-lis-N l-l are coupled by means of the azide methoddescribed in previous examples. After evaporation of the DMF, theresidue is taken up in aqueous ethyl acetate and the organic phasewashed with water and bicarbonate (5 percent).

The organic phase is distilled off, after which the residue isrecrystallised from ethyl acetate/alcohol/ether.

Peptide: Rf in Bz:EtOH (8:2)

D. Desamino-Met-Glu(OtBu)His-PheLys(- Boc)-Tra In the same manner asdescribed in C. the hydrazide DesaminoMet-Glu( OtBu )HisN 1-l obtainedfrom the corresponding methylester (ll B) is coupled toH-PheLys(Boc)-Tra (B) by means of the azide method previously described.Rf in BzzEtOl-l (8:2) =0.45 on SiO- E. Removal of protecting groups fromthe peptides ob tained in C and D 1n the manner described in example11.6 the above peptides are deprotected by means of TFA, after which theTFA molecules of the peptide salt thus obtained is exchanged for aceticacid molecules by means of an ionexchanger in the acetate form (e.g.Dowex X-8 in acetate form).

Peptide H-Met-Glu-His-PheLys-Tra 0 H-D-Met-Glu-His Phe-Lys-Tra 0.H-B-AlaClu-His-Phe-Lys-Tra 0. Desamino-Met-GluHis-Phe-Lys-Tra 0 F.Sulfoxides 45 mg of one of the peptide derivatives, obtained in E, aredissolved in 2.5 ml of acetic acid, after which 15 pl of 30 percenthydrogen peroxide are added. The mixture is treated in the mannerdescribed in example 1. The residue is taken up in butanol/water (1:2)and lyophilised.

(4:33/4: H4: 1) on SiO EXAIVIPLE XV Synthesis of HAGlul-lisPheArgTrpderivatives A. Boc-Arg(NO )TrpONB BocArg(NO )--OH (5.76 g) are dissolvedin ml of DMF and 2.52 ml of triethylamine. The solution is cooled downto 10 C, after which 2.38 ml of isobutyl chloroformate are added and themixture is stirred for 10 minutes at 10 C. To this solution a solutionof 6.14 g of HTrpONB.HCl in 40 ml of cooled DMF and 3.01 ml oftriethylamine are added.

The mixture is stirred for 30 minutes at l0 C, for 3 hours at 0 C andfor 20 hours at 20 C, after which the solvent is evaporated in vacuumand the residue taken up in ethyl acetate/water (1:1). The organic phaseis washed with water. 5 percent sodium bicarbonate and water, dried andevaporated to dryness in vacuum. Yield: 8.5 g of oil. Rf in Bz:EtOH(8:2) =0.79 (SiO B. HArg(NO )TrpONB 1 gram of the peptide prepared in Ais dissolved in 20 ml of methylene chloride, after which hydrogenchloride gas is bubbled through, while cooling. The solution is stirredfor 1 hour, after which the precipitate is filtered off and thoroughlywashed with dry methylene chloride. The precipitate was immediatelyprocessed further. Rf in AmzPyzWa (5:3:2) substance 0.93). C.ZPhe-Arg(NO )-Trp-ONB 3 grams of the dipeptide of B are dissolved in 20m1 of DMF. This solution is cooled down to C, after which 1.12 ml of TAAand 2.12 g of ZPheONP are added. The reaction mixture is stirred for 2hours at 0 C and for 20 hours at 20 C, and evaporated in vacuum.

= 0.66 on SiO (Rf starting The oily residue is dissolved in ethylacetate/water (2:1) and processed as described in A.

The organic phase is evaporated, the residue dissolved in 5 ml ofmethanol and poured slowly into 100 ml of dry ether to precipitate thepeptide.

Yield: 2.9 g; [(11 ==l5 (0 =1, DMFXR in BzzE- tOl-l (8:2) 0.48 on SiO 2grams of the above-mentioned peptide of C are dissolved in 40 ml ofacetic acid, after which 1 g of 10 percent palladium/charcoal is addedand the mixture hydrogenated for 2 days (Parr). After filtration theacetic acid is evaporated in vacuum, after which the residue is stirredinto dry ether. Yield: 98 percent. The slightly coloured foam is driedover solid potassium hydroxide. The substance contains 1.1 molecule ofacetic acid. Rf in Am:Py:Wa (523:2) =0.17 on SiO Of the tripeptide BocDMetGlu(Ot- Bu)l-lisN 1l (example IX.A) 1.17 g are dissolved in ml ofDMF. After the addition of 3 ml of 2 N HCl The organic phase isevaporated to dryness. The residue is stirred with dry ethylacetate/petroleum ether (9:1) and then dried.

Melting point: 214-2l7 (dec. Rf in Bu:Py:Ac:Wa (4:3/4: l/4:l) =0.48 onSiO G. In the same manner are prepared: RF"

1. Boc-Val-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.49 2.Boc-B-Ala-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.48 3.Boc-Ala-GIu(OtBu)-His-Phe-Arg-Trp-OH 0.52 4.Boc-(a-Me)Ala-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.54

5. Desamino-Met-Glu(OtBu)-His-Phe-Arg-Trp-OH 0.48

H. The peptides described in E, F and G are deprotected in EXAMPLE xviin THF at 0C the mixture is cooled to 20 C. Then 0.27 ml ifisoamyl-nitrite is added. The mixture is stirred for a few minutes,after which this azide solution is added to 8 ml of DMF, in which 2 mmolof the tripeptide of D (HPheArgTrpOH.acetate) have been dissolved. ThepH of the mixture is then adjusted to 7.3 with TAA. The reaction mixtureis stirred for 70 hours at 0 C, after which it is evaporated at reducedpressure, and the residue stirred into aqueous ethyl acetate.

Then the organic phase is washed with water, after which the organiclayer is dried. After filtration of this organic layer, petroleum etheris added to precipitate the peptide. Rf in Bu:Py:Ac:Wa (4:3/4zl/4z1)0.52 on SiO Of the tripeptide BocGlyGlu(Ot- Bu)HisN H- prepared fromBoc-GlyGlu(Ot- Bu)HisOMe by conversion with hydrazine hydrate, 2 mmolare coupled to the peptide HPhe-Arg-TrpOH in the manner described in E.The reaction mixture is stirred for 70 hours, after which it is pouredout' into 100 ml of ethyl acetate and extracted twice with saliferouswater.

ZPhe-ONP ester (2.1 g) is coupled to 2.24 g of H-Lys(Boc)Trp-OMe asdescribed in example XIILC. The oily ester is isolated and thensaponified with 1.1 equivalent of sodium hydroxide in 10 ml of methanol.On acidification the tripeptide acid precipitates, which isrecrystallised from methanol/water. Yield: 2.61 g. Rf in Bz:EtO1-l (8.2)=0.23 (SiO B. HPheLys(Boc)-TrpO1-l Hydrogenation of the tripeptidederivative of A gives a foam in quantitative yield. Crystallisation fromwater/methanol gives the tripeptide in 71 percent yield. Rf in BzzEtOl-l(8:2) =0.05 (SiO C. RGlu(OtBu)His-PheLys(Boc)Trp--OH (R Desamino-Met orBoc-DMet) I In the manner described in example VII, RGlu(Ot- Bu)-HisN1-1 in which R means: Desamino-Met or Boc-'-D-Met, is coupled to thetripeptide prepared in B by means of the azide method. The DMF solutionis poured into a tenfold quantity of water containing acetic acid (pH3-4) to obtain a precipitate. After filtration and stirring with waterthe peptide is isolated as an amorphous substance in 55-60 percentyield. D. RGlu--HisPhe-Lys-Trp-OH (R Desamino-Met or H-D-Met) I Cleavageof the protected peptides prepared in C, in the manner described inexample ILG, gives the acetate of the corresponding unprotectedpeptides. Rf in Bu:Py:Ac:Wa (4:3/4zl/4z1) 0.21 on SiO (for theH-DMet-peptide) and 0.25 (for the Desamino Met peptide). E. Sulfoxide ofR-Glu HisPhe-LysTrp-OH (R Desamino-Met or H-D-Met) In the mannerdescribed in example I the peptides obtained in D are oxidized in aceticacid containing 30 percent H Rfin Bu:Py:Ac:Wa (4:314:1/4: 1) =0.19 onSiO for the D Met( O) peptide and 0.22 for the Desamino-- Met( O)peptide.

EXAMPLE XVI] Sulfoxide of l-I-Met-Glul-lis-PheArg-Trp-OI-I, and ofH-MetGlul-IisPheLysTrp-Ol-I By oxidizing I-I-Met-Glu--HisPhe-Arg-Trp-OI-I and HMetGluI-IisPheLysTrp-OH with 30 percent hydrogenperoxide inacetic acid (see example I) the corresponding sulfoxides are obtainedwhich are purified via counter current. System in counter current:Bu:Ac:Wa (4: 1:5). Rf in BuzPyzAczWa (43/4: 1/4: 1 0.17 for thesulfoxide of the Lys-peptide and 0.20 for the Sulfoxide of theArg-peptide.

EXAMPLE XVIII Synthesis of A--Glu-l-Iis-PheArg-TrpGly peptides A.BocDMetGlu(OtBu)His-Phe-Arg-Trp-Gly-Ol-I In the manner described inexample IX.B the BocDMetGlu(OtBu)-HisN H is coupled toH-PheArgTrp-Gly-Ol-I.acetate. Rf in BuzPyzAczWa (423/411/4: l) 0.49 onSiO B. H-D-Met-Glu-I-Iis-Phe-Arg-Trp-Gly-OH Cleavage of the protectedpeptide obtained in A in the manner described in example II.G, gives theacetate of HD-MetGluI-IisPheArg-Trp-- GlyOH. Rf in BuzPyzAc2Wa(423/421/4z1) 0.16 on SiO C. Sulfoxide of HDMetGluHisPheArgTrp-Gly-OHThe peptide prepared in B is oxidized by means of acetic acid and 30percent H 0 in the manner described in example I and then lyophilised.Rf in Bu:Py:Ac:Wa (4:3/4z1/4z1) =0.14 on SiO D. In the same manner thefollowing peptides are prepared:

I-I--Gly-Glu-His-PheArgTrp-Gly-Ol-I H--/3AlaGlu-HisPheArgTrp-Gly-OHH-(aMe)Ala-Glu--HisPheArgTrp Gly-OH HValGlu--HisPheArgTrpGly-OHDesaminoMet-Glu-His-PheArg-Trp Gly-OH Desamino-Met(O)GluI-IisPhe-ArgTrp-Gly-OH EXAMPLE XIX Sulfoxide ofI-I-MetGlu-HisPheArg-Trp Gly-OH In the manner described in example I theheptapeptide H-Met-GluHis--PheArg-TrpGly-OH is oxidized and thenlyophilised. Rf in Bu:Py:Ac:Wa (423/411/421) =O.16 on SiO EXAMPLE XXSulfone of HMetGlu-HisPheLysTrp Gly-OH.acetate Of the peptideHMet-GluHisPheLys-Trp-GlyOH 200 mg are dissolved in a mixture of 0.5 mlof water, 0.1 m] of 4 perchloric acid, 0.02 ml of 0.5 M ammoniummolybdate, after which 0.06 ml of 30 percent hydrogen peroxide is added.

The mixture is stirred for 2 hours at a temperature of about 10 C. Thenthe mixture is stirred with Dowex X-S in the acetate form, after whichthe mixture is fil' tered and the filtrate lophilised.

Rf in Bu:Ac:Wa (4:1:1) =0.2l on SiO In the same manner were prepared:

p-Ol-l desamino-Met( O )Glu--HisPheOH.

EXAMPLE XXI Preparation of HMet(d, 0)GluHisPheAr- 26 grams ofL-methionine are oxidized to the corresponding Sulfoxide according tothe procedure of Toennies and Kolb cf. Biol. Chem. 128, 399 (1939),yield: 24 g, m.p. 250 C (dec). Both diastereomeric sulfoxides areresolved following the method as described by Lavine, J. Biol. Chem.169, 477 (1947).

Yield:

L-methionine(d, O) 8.7 g [011 110 (c 1,

H O) L-methionine(l, O) 1.8 g [01],, (c 1.

H O). These isomers were then convertedto the Bocprotected derivativesby means of Bee-N in a conven' tional manner:

42 (0 1, DMF) Boc-Met( l, O)Ol-I m.p. 68 C [(11 58 (c l, DMF) andthereupon convened into the corresponding N- hydroxy succinimide esterby treatment with DCCl and HOSu. These active ester derivatives are usedat once for the following coupling reaction. B. H-Met(d,O)GluI-IisPheArgTrp-- Gly-OH 929 Boc-Met(dsulfoxide)OSu are dissolved in20 ml DMF. The solution is cooled to 0 C whereupon is added a cooledsolution of 2.38 g of HGlu(OtBu I-Iis--PheArgTrp-Gly-OH and 0.83 ml ofN- ethyl morpholine in 25 ml of DMF.

The reaction mixture is stirred for 3 hours at 0C, 70 hours at roomtemperature and then evaporated. The residue is stirred with 40 ml ofethyl acetate and 10 ml of water, filtered, washed with petroleum etherand dried.

Yield: 2.1 g; Rf0.44 (BuzPyzAczWa 4:3/411/4:l) on SiO 2 grams ofBoc-Met(d- Sulfoxide )Glu( OtBu )I-Iis-PheArg-Trp GlyOH are treated with20 ml of percent TFA for 30 minutes. The solution is then stirred into300 ml of ether whereupon the precipitate is filtered off, washed withether and dried over solid potassium hydroxide. The trifluoro acetate isdissolved in 50 ml of tbutanol/water (1:1) and converted into theacetate by treatment with Dowex X-8 in the acetate form. Afterfiltration the filtrate is lyophilised.

HLMet HDMet O2), desamino-Met, desarninoMet O), desamino-Met 0 and themoiety H NBCO-, in which B is alkylene having l-6 carbon atoms, and inwhich X is selected from the group consisting of hydroxy, (N-

phenylalkyl-)-amino of the formula in which R, is selected from thegroup consisting of hydrogen and hydroxy and Alk is alkylene with l-6carbon atoms, and the group LPheY in which Y is selected from the groupconsisting of hydroxy and (N- aminoalkyl)-amino selected from the groupconsisting of descarboxy-lysyl and descarboxy-arginyl, the groups LLys-Zand LArg-Z, in which Z is selected from the group consisting of hydroxy,the group LTrp-OH, the group L- Trp-GlyOH, and a (N3- indolylethyl)amino group, and functional derivatives of said peptide selected fromthe group consisting of pharmaceutically acceptable acid addition salts,derivatives in which one or more free amino groups are substituted byacyl derived from an aliphatic carboxylic acid with l-6 carbon atoms,unsubstituted amides or 26 lower alkyl (l-6C) substituted amides ofthose peptides having a free carboxyl group, esters derived fromaliphatic or phenylaliphatic alcohols with l-l8 carbon atoms, and metalcomplexes thereof. 2. A peptide according to claim 1 of the formula:

in which A, is selected from a sulfoxide and a sulfone of H-Met and Xhas the meanings indicated in claim 1.

3. A peptide according to claim 1 of the formula:

A LGlu-L--His-X in which A is selected from v desamino-Met, desaminoMet(O) and desamino-Met( 0 and X has the meanings indicated in claim 1.

4. A peptide according to claim 1 of the formula:

in which A, stands for B-Ala and X has the meanings indicated in claim1.

7. Metal complexes of the peptides claimed in claim 1.

1. A PEPTIDE OF THE FORMULA:
 2. A peptide according to claim 1 of theformula: A1-L-Glu-L-His-X in which A1 is selected from a sulfoxide and asulfone of H-Met and X has the meanings indicated in claim
 1. 3. Apeptide according to claim 1 of the formula: A2-L-Glu-L-His-X in whichA2 is selected from desamino-Met, desamino-Met( -> O) and desamino-Met(-> O2) and X has the meanings indicated in claim
 1. 4. A peptideaccording to claim 1 of the formula: A3-L-Glu-L-His-X in which A3 standsfor Beta -Ala and X has the meanings indicated in claim
 1. 5. A peptideaccording to claim 1 of the formula:A-L-Glu-L-His-L-Phe-L-Lys-(N-3-indolyl ethyl)amine in which A has themeanings indicated in claim
 1. 6. A peptide according to claim 1 of theformula: A-L-Glu-L-His-L-Phe-L-Arg-(N-3-indolyl ethyl)amine in which Ahas the meanings indicated in claim
 1. 7. Metal complexes of thepeptides claimed in claim 1.